The use of gelatin in medical applications is well known. Gelatin is used because of its low immunogenic or antigenic properties. A disadvantage of the currently used animal gelatins is that it may be contaminated with other components from the bone or hide-matrix from which it is extracted like proteins or for example prions, causing Bovine Spongiform Encephalitis (BSE).
Various methods for producing collagen-like polypeptides such as gelatin in recombinant hosts such as bacteria, plants, insects, mammalian cells or yeasts are known.
WO 01/34646 describes production of collagen-like polypeptides in a variety of hosts and contains numerous suggestions for possible modifications of such polypeptides without teaching however how such modifications can be achieved. Glycosylation is mentioned as one of several post-translational processes that might be altered but no such alterations or means for achieving such alterations are proposed.
EP 0 926 543 describes production of recombinant collagen-like polypeptides in Pichia pastoris with high efficiency, but is silent with respect to glycosylation.
US 2003064436 describes hydroxylation as a posttranslational modification when expressing proteins in yeasts, but does not discuss glycosylation as a posttranslational modification.
Glycosylated aminoacids play an important role in (auto)immune reactions against collagen. It is suggested that they play role in T-cell recognition or T-cell binding. In human collagen N-linked glycosylation occurs on asparagine, and O-linked glycosylation on the —OH groups of hydroxylysines, serine and threonine.
It is also known that glycosylation of recombinant collagen-like polypeptides in non-human hosts differs from that in mammalian cells. The conditions under which aminoacids are glycosylated, such as the type of adjacent aminoacids, can be different, as well as the mechanism of glycosylation and thus the type of sugars attached.
Bretthauer and Castellino (Biotechnol. Appl. Biochem. (1999) 30, 193-200) describe glycosylation in the methylotrophic yeast Pichia pastoris.
Glycosylated aminoacids can also undergo phosphorylation of the sugar groups. Patents describing recombinant production of collagen-like proteins in various hosts, for example WO 01/34646 or EP 1 398 324, are silent with respect to this posttranslational modification. Phosphorylation of glycosylated aminoacids can lead to undesired high acidity of the polypeptide.